DESCRIPTION: Heme (iron-protoporphyrin IX) is the prosthetic group of proteins which play a key role in many biological processes including oxidative metabolism, the synthesis and sensing of diatomic gases, cellular differentiation, xenobiotic detoxification, gene regulation at the level of transcription, protein translation and targeting, and protein stability. Humans utilize heme as a source of iron from red meat because heme is more readily absorbed than inorganic iron in the intestine. In eukaryotes, heme is synthesized within the mitochondria. Free heme is insoluble in aqueous milieu and is cytotoxic due to peroxidase activity. Thus, a prima facie argument can be made that within cells specific pathways exist for the trafficking of heme from site of synthesis to various intracellular destinations for the incorporation of heme into apo-hemoproteins. Heme is synthesized via a multistep biosynthetic pathway with well-defined intermediates that were thought to be highly conserved through out evolution. The long-term objective of these studies is to define the molecular and cellular determinants of nutritional heme homeostasis in mammalian systems. The proposed studies will utilize C. elegans as a genetic model organism to elucidate pathways for heme uptake, sequestration, trafficking, and incorporation into hemoproteins. The biochemical mechanisms for heme acquisition in C. elegans will be evaluated by microscopy using fluorescent heme analogs, pulse-chase analysis, and heme uptake and incorporation assays using metabolic labeling with [59Fe]heme. The molecular and cellular pathways for heme trafficking in C. elegans will be identified by screening and classifying mutants that have heme-dependent defects in normal growth and development. The mutants will be charaterized biochemically, and the mutations will be mapped and localized by genetic recombination and SNP mapping. The results from these studies will provide new mechanistic insights into heme homeostasis in eukaryotes and may aid in the development of heme-based nutritional interventions for human iron deficiency, and permit design of novel drug targets to modulate xenobiotic metabolism.